Inhibition of protein kinase c alpha for treatment of diabetes mellitus and cardiovascular diseases

ABSTRACT

The invention relates to the use of agents impeding the expression and/or activity of protein kinase C-alpha (PKC-α), especially for treatment of patients with diabetes and complications such as diabetic nephropathy, retinopathy or neuropathy.

The present invention relates to the use of agents which reduce orinhibit the expression and/or activity of protein kinase C-α (PKC-α) forthe treatment and/or prevention of coronary heart disease, myocardialinfarction, peripheral occlusive disease, stroke, renal diseasesinvolving proteinuria, diabetic late effects and/or cardiovascularcomplications in patients with diabetes mellitus, cardiovascularcomplications in patients with hypertension, and cardiovascularcomplications in patients with hypercholesterolemia.

Diabetes mellitus is one of the most frequent diseases in the Westernworld and afflicts about 5% of the population. Diabetes mellitus issubdivided into diabetes type I, which usually occurs already in theyouth, and diabetes type II, which is also referred to as adult-onset ormaturity-onset diabetes. Due to a disorder in the glucose metabolism,permanently increased blood glucose levels occur in both diabetes forms,which results in different complications in the afflicted patients afterseveral years. The most frequent and at the same time most fearedcomplications are diabetic retinopathy, which results in blindness,diabetic neuropathy, which may lead to foot or leg amputations, anddiabetic nephropathy.

Diabetic nephropathy will develop in about 40% of all diabetes patientsand is the most frequent cause of chronic renal failure and dialysistreatment worldwide. About 30 to 40% of all new dialysis patientsexhibit diabetic nephropathy. Since diabetic renal damage developsslowly, the early identification of patients who have an increased riskof developing renal insufficiency is of great clinical importance forinitiating suitable therapeutic steps. One of the first clinical signsof beginning renal damage is the occurrence of a so-calledmicroalbuminuria. This involves the excretion of 30-300 mg of albumin in24 hours collected urine. Normally, less than 30 mg of albumin isexcreted per day. Under the current therapeutic conditions,microalbuminuria will occur in about 25% of diabetics with diabetes typeI or type II (Alzaid, Diabetes Care, 19: (1996), 79-89; Klein et al.,Diabetes Care, 22 (1999), 743-751; Valjnadrid et al., Arch. Intern.Med., 160 (2000), 1093-1100). The risk that renal insufficiency developsis about 10 times higher in patients with microalbuminuria as comparedto patients with normal albumin excretion. Diabetic nephropathy, whichis characterized by a proteinuria of more than 300 mg/day and/orrestricted renal function, will develop in about 5 to 10% of allpatients with diabetes and microalbuminuria per year. The risk thatdiabetic retinopathy will develop is also significantly increased indiabetics with microalbuminuria as compared to diabetics withoutmicroalbuminuria (Vigstrup and Mogensen, Acta Ophthalmol. (Copenh), 63(1985), 530-534).

As shown in long-term studies with more than ten years of follow-up,cardiovascular mortality is increased about twice in type II and type Idiabetics already in the stage of microalbuminuria as compared todiabetics without microalbuminuria, also after correction forconventional risk factors, such as cholesterol and hypertension (Rossinget al., Bmj, 313 (1996), 779-784; Gerstein et al., Diabetes Care, 23(2000), Suppl. 2: B35-39; Valmadrid et al., 2000). An increasedcardiovascular mortality can also be detected in patients withmicroalbuminuria without diabetes mellitus (Gerstein et al., Jama, 286(2001), 421-426).

There are various hypotheses of why microalbuminuria is an extremelyimportant marker for the development of complications in patients withdiabetes. According to the so-called “steno hypothesis” (Deckert,Feldt-Rasmussen et al., Diabetologia, 32 (1989), 219-226), the loss ofnegatively charged, i.e., anionic, molecules in the extracellular matrixis responsible for the formation of albuminuria, diabetic retinopathyand cardiovascular complications, for example, coronary heart disease.This hypothesis is supported by data acquired in both humans and animalmodel systems, and has been confirmed in recent years by resultsobtained by other working groups.

In the kidney, the urine is secreted in the renal corpuscles, theso-called glomeruli. To prevent the passage of proteins, for example,albumin, the blood side is separated from the urine side by a membranereferred to as the basal membrane. The basal membrane has small poreswhich allow smaller molecules to pass through the basal membrane whileprotein molecules cannot pass the membrane due to their size. Inpatients with microalbuminuria, the passage of small proteins, such asalbumin, nevertheless occurs although the pore size is not increased atfirst. To account for this phenomenon, it could be shown that moleculeswith negative charge which repel the also negatively charged proteinsare present in the pores or at the edge of the pores (Kverneland,Feldt-Rasmussen et al., Diabetologia, 29 (1986), 634-639; Deckert,Feldt-Rasmussen et al., Kidney Int., 33 (1988), 100-106; Kverneland,Welinder et al., Diabetologia, 31 (1988), 708-710). These molecules withnegative charges are proteoglycans. Proteoglycans are complexmacromolecules which consist of proteins to which polysaccharide chainsare covalently associated. The polysaccharide chains predominantlyconsist of heparan sulfate and have a high negative charge. Theproteoglycan which occurs most frequently in the body is perlecan.Perlecan is a protein of 460 kD and has several polysaccharide sidechains (Murdoch and Iozzo, Virchows Arch. A. Pathol. Anat. Histopathol.,423 (1993), 237-242; Iozzo, Cohen et al., Biochem. J., 302 (1994),625-639; Murdoch, Liu et al., J. Histochem. Cytochem., 42 (1994),239-249). In patients with diabetes and microalbuminuria, heparansulfate is hardly present in the glomerular basal membrane. Also inpatients with advanced diabetic nephropathy, heparan sulfate can nolonger be detected in the basal membrane, even though the protein chainsare still present. This effect is accounted for by the fact that heparansulfate synthesis is reduced under hyperglycemic conditions, as occur indiabetics (Parthasarathy and Spiro, Diabetes, 31 (1982), 738-741;Deckert, Feldt-Rasmussen et al., 1988; Nakamura and Myers, Diabetes, 37(1988), 1202-1211; Nerlich and Schleicher, Am. J. Pathol., 139 (1991),889-899; Makino, Ikeda et al., Nephron, 61 (1992), 415-421; Scandlingand Myers, Kidney Int., 41 (1992), 840-846; Vernier, Steffes et al.,Kidney Int., 41 (1992), 1070-1080; Tamsma, van den Born et al.,Diabetologia, 37 (1994), 313-320; Iozzo and San Antoniom, J. Clin.Invest., 108 (2001), 349-355). Further, it could be shown that heparansulfate proteoglycans not only prevent the glomerular filtration ofalbumin by their negative charge, but are probably also responsible forthe integrity of the pore size within the basal membrane (Deckert,Kofoed-Enevoldsen et al., Diabetologia, 36 (1993), 244-251). Thus, asthe renal insufficiency proceeds, the loss of heparan sulfateproteoglycans results in a destruction of the microstructure of thebasal membrane. These changes could explain why a great non-selectiveproteinuria with loss of larger proteins, such as immunoglobulin, occursin the course of diabetic nephropathy. Heparan sulfate proteoglycans arealso strong inhibitors of mesangial expansion in the renal corpuscle.This is of great interest since an expansion of the mesangial connectivetissue classically occurs in diabetes patients. Therefore, it is notsurprising that the loss of heparan sulfate proteoglycan in diabetespatients is accused as an important cause of mesangial expansion.

However, the loss of heparan sulfate in diabetics occurs not only in thekidney, but in almost all other organs. Thus, there is a clear reductionof heparan sulfate in the connective tissue of the retina, the skeletalmuscle, the arterial walls and the skin as well as on red blood cells.Endothelial cells also exhibit a reduced synthesis of heparan sulfate(Yokoyama, Hoyer, et al., Diabetes, 46 (1997), 1875-1880; van der Pijl,Daha et al., Diabetologia, 41 (1998), 791-798). Since heparan sulfateproteoglycans have important antithrombotic properties, the loss ofheparan sulfate proteoglycans can contribute to the formation ofmicrothrombi, for example, in the retinal vessels, and thus promote theformation of diabetic retinopathy (Marcum, Fritze et al., Am. J.Physiol., 245 (1983), H275-33; Marcum, McKenney et al., J. Clin.Invest., 74 (1984), 341-350; Marcum and Rosenberg, Biochemistry, 23(1984), 1730-1737; Marcum, Atha et al., J. Biol. Chem., 261 (1986),7507-7517). Further important anti-atherosclerotic functions of heparansulfate proteoglycans (HSPG) include the inhibition by HSPG of theproliferation of vascular smooth muscle cells, which results in theformation of arterial vascular lesions. HSPGs further inhibit thebinding of monocytes (inflammatory cells) to the subendothelialconnective tissue. HSPGs also inhibit the subendothelial binding anddeposition of lipoprotein a and oxidize LDLs, which play a critical rolein the formation of atherosclerosis. HSPGs are also important regulatorsin angiogenesis, i.e., in the formation of new vessels in damaged bodyregions (Rosenberg, Shworak et al., J. Clin. Invest., 100 (1997), p.67-75; Pillarisetti, Trens Cardiovasc. Med., 10 (2000), 60-65; Iozzo andSan Antonio, 2001). Therefore, the loss of heparan sulfate proteoglycanis important not only to the development of diabetic nephropathy anddiabetic retinopathy, but also in the development of cardiovascularcomplications.

A further aspect is the fact that microalbuminuria will occur inpatients with hypertension. To date, this phenomenon has been explainedby an increased pressure in the renal corpuscles, assuming that albuminis increasedly secreted. However, if this is the case, it must beconsidered that patients with a constantly high arterial blood pressurealso have a high cardiovascular risk, irrespective of whether theyexhibit microalbuminuria. However, this is not the case, as could beshown in several prospective studies. Hypertensive patients withmicroalbuminuria show a cardiovascular morbidity and mortality which isabout twice as high as that of similarly hypertensive patients withotherwise comparable risk profile, for example, hypercholesterolemia,smoking history and diabetes (Sleight, J. Renin Angiotensin AldosteroneSyst., 1 (2000), 18-20; Crippa, J. Hum. Hypertens., 16 (Suppl. 1)(2002), p. 74-7; Diercks, van Boven et al., Can. J. Cardiol., 18 (2002),525-535). Accordingly, microalbuminuria is an independent risk parameterof the development and prognosis of cardiovascular diseases. This can beexplained only by the fact that a change in the whole vascular systemoccurs in patients with microalbuminuria. However, to date, it has beenunclear which disorder in patients with hypertension is the basis ofmicroalbuminuria.

The object of the invention is to provide agents which can be employedfor the therapy of microalbuminuria, especially in patients withdiabetes mellitus and patients with hypertension in order to treatand/or prevent the late effects associated with diabetes, especiallydiabetic retinopathy, diabetic nephropathy and diabetic neuropathy, andcardiovascular complications as well as the cardiovascular complicationsassociated with hypertension.

The present invention achieves this object by using agents which reduceor inhibit the expression and/or activity of protein kinase C-α (PKC-α)for the treatment and/or prevention of vascular diseases, cardiovasculardiseases, renal diseases involving proteinuria, diabetic late effectsand/or cardiovascular complications in patients with diabetes mellitus,cardiovascular complications in patients with hypertension, and/orcardiovascular complications in patients with hypercholesterolemia.

In the prior art, it has been supposed to date that the β2 isoform ofprotein kinase C is responsible for the development of the diabeticcomplications. On the one hand, the β2 isoform is produced at anincreased level in the tissue of diabetic animals (Inoguchi et al.,Proc. Natl. Acad. Sci. USA, 89 (1992), 11059-11063), and on the otherhand, the protein kinase C-p specific inhibitor LY333531 results in areduced proteinuria as a sign of reduced renal damage in rodents withtype I and type II diabetes (Ishii et al., J. Mol. Med., 76 (1998),21-31; Koya et al., Faseb J., 14 (2000), 439-447).

Protein kinase C-α “knock out” mice produced according to the inventionwhich are not able to form protein kinase C-α surprisingly did notdevelop albuminuria after the induction of diabetes by means ofstreptozotozin. In contrast, control animals which were geneticallysubstantially identical except for the change of protein kinase C-αexpression developed a clear albuminuria. According to the invention,the further examination of the “knock out” animals showed completelysurprisingly that the animals were able to form heparan sulfate at anormal level under diabetic conditions. In contrast, the control animalswere hardly able to form heparan sulfate any longer under diabeticconditions.

Histological examinations performed according to the invention resultedin further significant changes in the protein kinase C-α “knock out”mice. According to the invention, using immunohistochemical methods, itcould be shown that the lack of protein kinase C-α entrains furthersignificant differences in the expression of VEGF (vascular endothelialgrowth factor) and the related receptor (VEGF-R II). While a significantincrease of the expressed amounts of VEGF and VEGF-R II receptor couldbe detected in diabetic control animals, a significantly lower increaseof the expressed amounts of VEGF and VEGF-R II receptor was establishedin the protein kinase C-α “knock out” animals. This result is of immenseimportance because increased VEGF expression is considered one of themost important mediators for the development of diabetic retinopathy(Aiello and Wong, Kidney Int. Suppl., 77 (2000), p. 113-9; Benjamin, Am.J. Pathol., 158 (2001), 1181-1184).

From the results according to the invention, it can be seen that proteinkinase C-α plays a key role in the regulation of the heparan sulfateproteoglycan formation and in the manifestation of proteinuria. Theresults according to the invention also show that protein kinase C-αplays a significantly more important role in the manifestation ofproteinuria as compared to protein kinase C-α, wherein protein kinaseC-β is evidently capable of taking over at least part of the functionsof protein kinase C-α, however. The results according to the inventionfurther show that an inhibition of the protein kinase C-α isoformselectively offers protection from both the development of diabetic lateeffects, such as diabetic nephropathy, diabetic retinopathy and/orcardiovascular complications, and the development of diseases which areaccompanied by proteinuria.

Thus, according to the invention, there is provided the use of agentswhich reduce or inhibit the expression and/or activity of protein kinaseC-α (PKC-α) for the treatment and/or prevention of vascular diseases,cardiovascular diseases, renal diseases involving proteinuria, diabeticlate effects and/or cardiovascular complications in patients withdiabetes mellitus, cardiovascular complications in patients withhypertension and/or cardiovascular complications in patients withhypercholesterolemia.

In the context of the present invention, “diseases” refers to disordersof the vital processes in organs or in the whole organism which resultin subjectively felt or objectively detectable physical, psychic ormental changes. “Complications” or “late effects” means consequentialdiseases or secondary diseases, i.e., a second disease which occurs inaddition to a primary clinical picture.

According to the invention, the diseases to be treated are, inparticular, vascular diseases, cardiovascular diseases, renal diseasesinvolving proteinuria, diabetes mellitus with and without associatedlate effects and/or cardiovascular complications, hypertension with andwithout associated cardiovascular complications and/orhypercholesterolemia with and without associated cardiovascularcomplications.

In the context of the present invention, “vascular diseases” means, inparticular, diseases of the arteries which may lead to functional ororganic circulatory disturbance. In a preferred embodiment of theinvention, the vascular disease is a peripheral arterial occlusivedisease. “Arterial occlusive disease” means a disease which is caused bystenosing or obliterating changes in the arteries and results incirculatory disturbance with ischemia in tissues or organs which dependon supply. Diabetes mellitus, in particular, results in chronicocclusive diseases which are caused, inter alia, by obliteratingatherosclerosis and also angiopathies and angioneuropathies.

“Cardiovascular diseases” means diseases and disorders which affect thefunction of the heart and circulation, for example, the filling stateand tonus of the circulatory system, the output performance of theheart, the neural and humoral coupling mechanisms between the heart andcirculation etc. In a preferred embodiment of the invention, thecardiovascular diseases are coronary heart disease, myocardialinfarction and stroke.

“Coronary heart disease” means the clinical manifestation of a primarycoronary insufficiency in which the constriction or occlusion ofcoronary vessels results in a reduction of circulation and thus thesupply of energy-delivering substrates and oxygen to the cardiac muscle.

“Myocardial infarction” means the necrosis of a localized region of thecardiac muscle which mostly occurs acutely as a complication in chroniccoronary heart disease. The cause of myocardial infarction is acontinuing critical circulation deficiency in coronary insufficiency andextended coronary spasms, especially in the region of a pre-existingeccentric coronary stenosis. A myocardial infarction is often manifestedupon physical or psychic stress as a consequence of an increased oxygendemand of the cardiac muscle or upon an acute interruption of the bloodsupply.

“Stroke” or “apoplexy” means an ischemic cerebral infarction as aconsequence of arterial circulation disorders of the brain. Stroke iscaused by embolisms derived from atherosclerotic changes in extracranialvessels or from the heart, less frequently as a consequence of stenosisor cerebral microangiopathies.

In the context of the present invention, “renal diseases involvingproteinuria” means, in particular, parenchymal kidney diseases which arecharacterized by the presence of proteins in the urine. The proteinuriamay be glomerular proteinuria, tubular proteinuria or mixedglomerulo-tubular proteinuria. The exclusively renal excretion ofalbumin and transferrin characterizes the selective proteinuria whichoccurs, for example, in minimal-change nephropathy. In non-selectiveproteinuria, IgG can also be detected in the urine. This form ofproteinuria can be found, for example, in renal amyloidosis, but also inan advanced state of diabetic nephropathy. Tubular proteinuria is basedon tubolo-interstitial diseases which affect reabsorptive processes,which results in the excretion of low-molecular weight proteins.Clinically, tubular proteinuria is of importance, in particular, whenassociated with other defects of the proximal tubule. Tubularproteinurias occur, inter alia, in diseases such as hereditarytubulopathy, renal-tubular azidosis, interstitial nephritis induced bybacteria or medicaments, acute renal failure, heavy metal poisoning,Bence-Jones nephropathy and in the postsurgical phase of kidneytransplantation. Mixed glomerulo-tubular proteinuria is often based onprimary glomerular diseases with pronounced secondary interstitialchanges.

Therefore, in a preferred embodiment of the invention, the renaldiseases involving proteinuria are, in particular, minimal-changenephropathy, other glomerulopathies, kidney amyloidosis, hereditarytubulopathy, renal-tubular azidosis, interstitial nephritis induced bybacteria or medicaments, acute renal failure, Bence-Jones nephropathyand the postsurgical phase of kidney transplantation.

In the context of the present invention, “diabetes mellitus” meansvarious forms of glycose metabolic disorders with different etiologiesand symptoms. A common characteristic is a relative or absolute insulindeficiency. Diabetes mellitus diseases are characterized by a permanentincrease of blood glucose level (hyperglycemia) or by a mistimedutilization of supplied glucose. Diabetes mellitus is subdivided intotype I (insulin-dependent; IDDM) and type II (non-insulin-dependent;NIDDM).

Diabetes-specific and diabetes-associated chronic complications includemicroangiopathy, such as retinopathy, nephropathy and neuropathy,polyneuropathy, diabetic foot, disorders of the skeletal, supporting andconnective tissue as well as macroangiopathy, especially coronary heartdisease, cerebral circulation disorder and peripheral arterial occlusivedisease.

“Diabetic retinopathy” means a microangiopathy of the eye-groundoccurring in diabetes mellitus. Forms of diabetic retinopathy arenon-proliferative retinopathy (background retinopathy), such as retinalhemorrhages, microaneurysms, hard exudates, retinal edema with loss ofvisual acuity, as well as proliferative retinopathy, in which there isadditional occurrence of cotton-wool spots and angioneogenesis on and infront of the retina with vitreous hemorrhage due to retinal ischemiafrom vascular occlusion. Proliferative retinopathy may result intraction retinal detachment, neovascular glaucoma and blindness.

In the context of the present invention, “diabetic nephropathy”, whichis also referred to as diabetic glomerulosclerosis, means damage to theglomerular kidney capillaries. Clinically, diabetic nephropathy ismanifested by proteinuria, hypertension, edemas, a diffuse widening ofthe basal membrane, mesangial hypertrophy and later nodular swellings inthe loops of the glomerulus with constriction of the vascular lumen aswell as fibrinoid depositions in the capillary wall and microaneurysms.

In the context of the present invention, “diabetic neuropathy” means adisease of the peripheral nerves. In particular, it means symmetricdistal sensomotoric polyneuropathy and autonomous neuropathy. Peripheralneuropathy is typically manifested in the lower extremities, beginningwith the feet, and proceeds towards proximal and not infrequently alsoaffects the arms. The symptoms vary significantly, and complaints suchas pain, numbness and paresthesia often result in exacerbation.

According to the invention, “cardiovascular complications in diabetes”means cardiovascular and vascular diseases, especially peripheralocclusive disease, coronary heart disease, myocardial infarction andstroke, which occur as a consequence of diabetes mellitus.

In the context of the present invention, “hypertension” means high bloodpressure or hypertensive heart disease which is characterized by apermanent increase of blood pressure to values of more than 140 mm Hgsystolic and more than 90 mm Hg diastolic. According to the invention,“cardiovascular complications associated with hypertension” meanscardiovascular and vascular diseases, especially peripheral occlusivedisease, coronary heart disease, myocardial infarction and stroke, whichoccur as a consequence of hypertension.

In the context of the present invention, “hypercholesterolemia” means anincreased cholesterol level in the blood, wherein thehypercholesterolemia may occur primarily or secondarily as a consequenceof diabetes. Hypercholesterolemia is a risk factor of atherosclerosis.According to the invention, “cardiovascular complications associatedwith hypercholesterolemia” means cardiovascular and vascular diseases,especially peripheral occlusive disease, coronary heart disease,myocardial infarction and stroke, which occur as a consequence ofhypercholesterolemia.

In the context of the present invention, a “protein kinase C” or “PKC”means a family of proteins which plays an essential role in signaltransmission, the PKC proteins serving intracellular regulatoryfunctions by the phosphorylation of substrates, such as enzymes,transcription factors and/or cytoskeleton proteins. For example,activation of the PKC proteins results in an activation of furtherprotein kinases including mitogen-activated protein kinase (MAPK), whichare thus substrates of the PKC proteins. Protein kinase C proteins arethe main phorbol ester receptors. The protein kinase C family ofproteins comprises at least twelve isoforms in mammal cells which aresubdivided into three different subfamilies. The so-called conventionalprotein kinase C isoforms (cPKC) comprise the isoforms PKC-α, PKC-βI andits splice variant βII as well as PKC-gamma. The so-called novel proteinkinase isoforms (nPKC) comprise the isoforms PKC-delta, PKC-epsilon,PKC-eta and PKC-theta. The so-called atypical protein kinase C isoforms(aPKC) comprise the isoforms PKC-zeta and PKC-lambda (also known asPKC-iota). Further isoforms are PKC-mu (also referred to as proteinkinase D) and the PKC-related kinases (PRK) which may be separatesubfamilies (Toker, Frontiers in Biosciences, 3 (1998), d1134-1147). ThePKC isoforms are distinguished in both their amino acid sequences andthe nucleic acid sequences coding for the amino acid sequences (Coussenset al., Sciences, 233 (1986), 859-866). The PKC proteins all have adomain structure. Their cellular expression patterns, their mechanismsof activation and their substrate specificities are also different.

The majority of protein kinase C isoforms are not membrane-bound beforeactivation and is diffusely distributed in the cytoplasm. The activationof the activity of each isoform by treating the cells with the phorbolcompound 12-O-tetradecanoylphorbol-13-acetate results inisozyme-specific changes of cell morphology as well as in a rapid andselective redistribution of the different PKC isozymes into differentsubcellular structures. The protein kinase C-α isoform becomes enriched,in particular, in the endoplasmic reticulum and at the cellular edge,while the PKC βII isoform is enriched in the actin-rich microfilamentsof the cytoskeleton. The substrate specificity of the PKC isoforms ismediated at least partially by the subcellular distribution of theactivated protein kinase C isozymes.

“Protein kinase C-α” means a protein which is activated by calcium ionsand diacylglycerol, the activated protein kinase C-α becoming enriched,in particular, in the endoplasmic reticulum and at the cellular edge.The amino acid sequence of PKC-α and the nucleic acid sequence codingfor PKC-α are described in Coussens et al., Sciences, 233 (1986),859-866. The PKC-α protein has a similar domain structure as theremaining cPKC proteins. The protein comprises a pseudosubstrate domain,a cysteine-rich region, a calcium-binding domain and a catalytic domain.PKC-α can be activated by diacylglycerol, phorbol ester,phosphatidylserine and calcium.

In the context of the present invention, “agents which reduce or inhibitthe expression of protein kinase C-α” means those agents whichcompletely prevent or at least reduce the synthesis of a functionalPKC-α protein under both in-vitro and in-vivo conditions, said reductionor inhibition concerning the transcription of the DNA sequence codingfor PKC-α into a complementary mRNA sequence, the processing of themRNA, the translation of the mRNA into a polypeptide chain, theprocessing of the polypeptide and/or posttranslational modifications ofthe polypeptide. Thus, the use of agents which reduce or inhibit theexpression of protein kinase C-α may cause that either no functional,for example, activatable, PKC-α protein is prepared at all, or that theamount of the produced functional, for example, activatable, PKC-αprotein is reduced. However, the use of agents which reduce or inhibitthe expression of protein kinase C-α may cause that a non-functional,for example, non-activatable, PKC-α protein or an only partiallyfunctional PKC-α protein is produced.

In the context of the present invention, “agents which reduce or inhibitthe activity of protein kinase C-α” means those agents which cancompletely or partially eliminate the biological activity of thefunctional PKC-α protein under both in-vitro and in-vivo conditions. Thecomplete or partial inactivation of the PKC-α protein may be effected,for example, by a direct interaction of the agent employed with thePKC-α protein. The direct interaction between the agent and PKC-αprotein can be effected, for example, by covalent or non-covalentbinding. The interaction between the agent and PKC-α protein may alsocause, for example, chemical changes in the protein kinase, whichresults in a loss of the biological activity of the protein kinase. Theinteraction may also lead, for example, to a specific degradation ofPKC-α. However, agents which reduce or inhibit the activity of proteinkinase C-α may also be those which modify or eliminate or bind tospecific substrates, target structures or target molecules of PKC-α insuch a way that the biological activity of PKC-α is reduced orcompletely suppressed. Agents which reduce or inhibit the activity ofprotein kinase C-α may also be those which prevent the translocation ofPKC-α into the endoplasmic reticulum or to the edge of the cell afteractivation, for example, activation by phorbol treatment, so that PKC-αcannot interact with its specific substrates, target structures ortarget molecules.

In a particularly preferred embodiment of the invention, the agentsemployed according to the invention are agents which specifically reduceor inhibit the expression and/or activity of PKC-α, but not theexpression and/or activity of other PKC isoforms, for example, PKC-β.

According to the invention, the agents which specifically reduce orinhibit the expression and/or activity of PKC-α are selected from thegroup consisting of nucleic acids which reduce or inhibit the expressionof the protein kinase C-α gene, vectors containing said nucleic acid,host cells containing said vectors, substances which inhibit or reducethe expression of protein kinase C-α, substances which inhibit thetranslocation of protein kinase C-α, antagonists of protein kinase C-αactivity, and inhibitors of protein kinase C-α activity.

According to the invention, the nucleic acid employed is preferablyselected from the group consisting of

-   -   a) a nucleic acid coding for human protein kinase C-α, or a        fragment thereof;    -   b) a nucleic acid which is complementary to the nucleic acid        according to a), or a fragment thereof;    -   c) a nucleic acid which is obtainable by substitution, addition,        inversion and/or deletion of one or more bases of a nucleic acid        according to a) or b), or a fragment thereof; and    -   d) a nucleic acid which has more than 80% homology with a        nucleic acid according to a) through c), or a fragment thereof.

In the context of the present invention, a “nucleic acid coding forprotein kinase C-α, or a fragment thereof” means a nucleic acid whichcodes for a PKC-α protein or a fragment thereof which comprises thefunctional domains, especially the pseudosubstrate domain, thecysteine-rich region, the calcium-binding domain and a catalytic domainof native protein kinase C-α. In a preferred embodiment of theinvention, the nucleic acid used according to the invention codes forhuman PKC-alpha or parts thereof.

In the context of the invention, “homology” means a sequence identity ofat least 80%, preferably at least 85% and more preferably more than 90%,95%, 97% and 99%. Thus, the term “homology” which is known to theskilled person designates the degree of relationship between two or morenucleic acid molecules, which is determined by the agreement between thesequences.

The nucleic acid used according to the invention may be a DNA or RNAsequence, especially in a linear form. The nucleic acid may be isolatedfrom natural sources, for example, from eukaryotic tissues, preferablymammal tissues, more preferably from human tissues, or preparedsynthetically.

According to the invention, it is provided, in particular, that thenucleic acid used as the agent, if inserted in a vector, especially anexpression vector, can inhibit the expression of the gene of humanprotein kinase C-α in a host cell in antisense orientation to apromoter. When the nucleic acid employed according to the invention isinserted in a vector in antisense orientation, i.e., when an antisenseconstruct of the nucleic acid employed according to the invention isemployed, the nucleic acid will be transcribed as an antisense nucleicacid. Then, when the native PKC-alpha gene of the cell is transcribed,the antisense transcript produced of the nucleic acid used according tothe invention can bind through Watson-Crick base pairing to the mRNAtranscript of the native protein kinase C-α gene which is in senseorientation to form a duplex structure. In this way, the translation ofthe mRNA of the native PKC-α gene into a polypeptide is selectivelysuppressed, and the expression of the native PKC-alpha is specificallyinhibited without inhibiting the expression of other cellular PKCisoforms.

In a preferred embodiment of the invention, it is provided that thenucleic acid used for the production of antisense constructs does notcomprise the entire sequence coding for PKC-alpha, but only fragmentsthereof. Such fragments comprise at least 10 nucleotides, preferably atleast 50 nucleotides, more preferably at least 200 nucleotides, whereinthe nucleotide regions of the sequence coding for PKC-alpha which arespanned by the fragments are selected in such a way that, when thefragments are expressed in antisense orientation in a cell, specificinhibition of the expression of PKC-alpha, especially human PKC-alpha,occurs, but not inhibition of other PKC isoforms, for example, thePKC-beta isoforms.

According to the invention, it is provided that the above mentionednucleic acid or the suitable fragment thereof is inserted in a vectorunder the control of at least one expression regulating element, whereinthe nucleic acid or its fragment is inserted in an antisense orientationwith respect to said expression regulating elements. Thus, after thevector has been introduced into a cell, for example, a mammal cell,especially a human cell, the nucleic acid or its fragment can beexpressed in antisense orientation and thus efficiently inhibit theexpression of the native PKC-alpha of the cell. Preferably, the vectoris a plasmid, cosmid, bacteriophage or virus.

Therefore, the present invention also relates to a vector whichcomprises a nucleic acid sequence coding for PKC-alpha or a fragmentthereof under the functional control of at least one expressionregulating element, wherein the nucleic acid or its fragment is insertedin an antisense orientation with respect to said expression regulatingelement. Said expression regulating element is, in particular, apromoter, a ribosome binding site, a signal sequence or a 3′transcription terminator.

Another embodiment of the invention relates to a host cell whichcontains an above described vector. In particular, the host cell is amammal cell, preferably a human cell. In a particularly preferred form,the human cell is an adult stem cell.

In a preferred embodiment of the invention, synthetically preparedantisense oligonucleotides which comprise at least 10 nucleotides,preferably at least 50 nucleotides, more preferably at least 200nucleotides, are employed for inhibiting the expression of PKC-alpha.Such antisense oligonucleotides can be directly employed for inhibitingPKC-alpha expression, i.e., need not be inserted into a vector andexpressed under cellular conditions. In a particularly preferredembodiment, these PKC-α-specific antisense oligonucleotides are theproduct ISIS 3521 from Isis Pharmaceuticals, which is a strong selectiveinhibitor of protein kinase alpha expression. In a further particularlypreferred embodiment of the invention, the PKC-α-specific antisenseoligonucleotides employed according to the invention are the antisenseoligodeoxynucleotides described by Busutti et al., J. Surg. Pathol., 63(1996), 137-142.

According to the invention, the above mentioned nucleic acids, thevectors containing such nucleic acids or the host cells containing suchvectors can be equally employed as agents for the treatment and/orprevention of vascular diseases, cardiovascular diseases, renal diseasesinvolving proteinuria, late effects and/or cardiovascular complicationsassociated with diabetes mellitus, cardiovascular complicationsassociated with hypertension, and/or cardiovascular complicationsassociated with hypercholesterolemia, for example, within the scope ofgene therapy.

In another preferred embodiment of the invention, it is provided that anactivator of protein kinase C-α is employed for inhibiting or reducingthe expression of protein kinase alpha. Preferably, said activator is aphorbol compound, especially 12-O-tetradecanoylphorbol-13-acetate (TPA)or phorbol-12,13-dibutyrate (PDBu). It is known that the incubation ofcells with, for example, PDBu over a period of 16 h to 24 h results in acomplete down regulation of PKC-alpha (Busutti et al., J. Surg. Res., 63(1996), 137-142). Also, it is known that treatment with a higher TPAconcentration, for example, 1.6 μM, completely inhibits PKC-alphaexpression. Therefore, according to the invention, the treatment ofafflicted tissues with phorbol esters in a concentration of preferablymore than 1.6 μM over a period of at least 15 h is provided in order toblock the expression of PKC-alpha in the respective tissues or organspartially or completely.

In another preferred embodiment, the use of an inhibitor for inhibitingor reducing the activity of protein kinase α is provided. In the contextof the present invention, “inhibitor” means a substance whichcompetitively inhibits the biological activity of protein kinase C-α,allosterically changes the spatial structure of PKC-α, or inhibits PKC-αby substrate inhibition.

In a preferred embodiment of the invention, the inhibitor is an antibodywhich specifically reacts with protein kinase C-α. “Antibody” meanspolypeptides which are essentially coded for by an immunoglobulin geneor genes or fragments thereof and which are able to specifically bindand recognize an analyte, i.e., an antigen. The binding of the antibodyto PKC-α inhibits the biological activity of the latter. According tothe invention, the antibodies against the protein kinase C-α may beemployed as intact immunoglobulins or as a number of fragments producedby cleavage with various peptidases. The term “antibodies” as usedaccording to the invention also relates to modified antibodies, forexample, oligomeric antibodies, reduced antibodies, oxidized antibodiesand labeled antibodies. The term “antibody” also comprises antibodyfragments prepared either by modifying the whole antibody, or de novowith the use of recombinant DNA methods. Therefore, the term “antibody”comprises both intact molecules and fragments thereof, such as Fab,F(ab′)₂ and FV, which can bind to the epitopic determinants. Theseantibody fragments retain the ability to bind selectively to thecorresponding antigen. Methods for the preparation of antibodies orfragments thereof are known in the prior art.

In a preferred embodiment of the invention, the antibody employedaccording to the invention for inhibiting the activity of protein kinaseC-α is a monoclonal or polyclonal antibody. According to the invention,the antibody may also be a humanized antibody. In a particularlypreferred embodiment of the invention, the antibodies used forinhibiting the activity of protein kinase C-α are those as described byGoodnight et al., J. Biol. Chem., 270 (1995), 9991-10001.

In a further preferred embodiment of the invention, it is provided thatthe inhibitor employed according to the application for inhibitingPKC-alpha changes the phosphorylation state of protein kinase C-α andthus inhibits or at least reduces the activity of PKC-alpha. FromTasinato et al., Biochem. J., 334 (1998), 243-249, it is known thatalpha-tocopherol can inactivate the cellular protein kinase C-alpha bychanging the phosphorylation state of PKC-alpha. Therefore, in aparticularly preferred embodiment of the invention, the use ofalpha-tocopherol for inhibiting the activity of PKC-alpha and thus forthe treatment and/or prevention of vascular diseases, cardiovasculardiseases, renal diseases involving proteinuria, diabetic late effectsand/or cardiovascular complications in patients with diabetes mellitus,cardiovascular complications in patients with hypertension and/orcardiovascular complications in patients with hypercholesterolemia isprovided.

In a further preferred embodiment of the invention, the use ofantagonists of PKC-alpha for the treatment and/or prevention of vasculardiseases, cardiovascular diseases, renal diseases involving proteinuria,diabetic late effects and/or cardiovascular complications in patientswith diabetes mellitus, cardiovascular complications in patients withhypertension and/or cardiovascular complications in patients withhypercholesterolemia is provided. In the context of the presentinvention, “antagonist” means a substance which competes with PKC-alphafor the binding to a PKC-alpha-specific substrate, but without causingthe same effect as PKC-alpha after binding to the substrate. The term“antagonists” also includes substances which are adapted to an inactiveconformation of a PKC-alpha-specific substrate due to their structureand therefore prevent the activation of the substrate by PKC-alpha.

In a preferred embodiment of the invention, a derivative of PKC-alphawhich can bind to the substrates of the native PKC-alpha, but withoutcausing the same biological effect as native PKC-alpha after bindingthereto, is employed as the antagonist for inhibiting the PKC-alphaactivity.

In the context of the present invention, “derivatives” means functionalequivalents or derivatives of protein kinase C-α which are obtained bysubstituting atoms or molecular groups or residues while retaining thePKC-α basic structure, and/or whose amino acid sequences differ from thenaturally occurring sequence of human or animal PKC-α molecules in atleast one position, but which essentially have a high degree of homologyon the amino acid level. According to the invention, the term“derivative” also includes fusion proteins in which functional domainsof another protein, for example, another PKC-α inhibitor, are present inthe N-terminal or C-terminal portions.

The differences between a derivative and native PKC-α may arise, forexample, from mutations, such as deletions, substitutions, insertions,additions, base exchanges and/or recombinations of the nucleotidesequences coding for the PKC-α amino acid sequences. Of course, thesemay also be naturally occurring sequence variations, for example,sequences from another organism or sequences mutated in a natural way,or mutations which have been purposefully introduced into thecorresponding sequences by usual means known in the art, for example,chemical agents and/or physical agents.

In another preferred embodiment of the invention, an analogue ofPKC-alpha is employed as the antagonist for inhibiting the PKC-alphaactivity. In the context of the present invention, “analogues” ofprotein kinase C means compounds which do not have an amino acidsequence identical with that of protein kinase C-α, but whosethree-dimensional structure is highly similar to that of protein kinaseC-α. The analogues of PKC-α employed according to the inventionpreferably have substrate specificity properties similar to those ofPKC-α, i.e., they can bind to the PKC-alpha-specific substrates, butpreferably lack the catalytic properties of PKC-α. Therefore, theprotein kinase C-α analogues employed according to the invention may be,for example, compounds which contain the amino acid residues responsiblefor the binding of protein kinase C-α to PKC-α substrates in a suitableconformation and are therefore able to mimic the essential properties ofthe binding region of protein kinase C-α, but without possessing thesame catalytic properties as protein kinase C-α.

In another embodiment of the invention, it is provided that, for thetreatment and/or prevention of vascular diseases, cardiovasculardiseases, renal diseases involving proteinuria, late effects and/orcardiovascular complications in patients with diabetes mellitus,cardiovascular complications in patients with hypertension, and/orcardiovascular complications in patients with hypercholesterolemia,agents are employed which reduce or inhibit not only the expressionand/or activity of protein kinase C-α (PKC-α), but at the same time theexpression and/or activity of protein kinase C-β (PKC-β). In J. Invest.Dermatol., 117 (2001), 605-611, Takahashi and Kamimura describe that theimmunosuppressant cyclosporine A reduces the expression of the proteinkinase C isoforms alpha, beta I and beta II at the same time. Therefore,in a particularly preferred embodiment of the invention, the use ofcyclosporine A for reducing the expression of PKC-alpha and PKC-beta andthus for the treatment of the above mentioned diseases is provided.

In another preferred embodiment of the invention, it is provided thatthe agent which specifically reduces or inhibits the expression and/oractivity of protein kinase C-α is used in combination with an agentwhich specifically reduces or inhibits the expression and/or activity ofprotein kinase C-β. According to the invention, “protein kinase C-β”includes both protein kinase C-βI and the splice variant βII.

According to the invention, it is provided that the agent which reducesor inhibits the expression and/or activity of protein kinase C-β isselected from the group consisting of nucleic acids which reduce orinhibit the expression of the protein kinase C-β gene, vectorscontaining said nucleic acid, host cells containing said vectors,substances which inhibit or reduce the expression of protein kinase C-β,substances which inhibit the translocation of protein kinase C-β,antagonists of protein kinase C-β activity, and inhibitors of proteinkinase C-β activity.

In a preferred embodiment of the invention, the nucleic acid to beemployed as an agent is selected from the group consisting of

-   -   a) a nucleic acid coding for human protein kinase C-β, or a        fragment thereof;    -   b) a nucleic acid which is complementary to the nucleic acid        according to a), or a fragment thereof;    -   c) a nucleic acid which is obtainable by substitution, addition,        inversion and/or deletion of one or more bases of a nucleic acid        according to a) or b), or a fragment thereof; and    -   d) a nucleic acid which has more than 80% homology with a        nucleic acid according to a) through c), or a fragment thereof.    -   In a preferred embodiment of the invention, the nucleic acid        used according to the invention codes for human PKC-alpha or        parts thereof.

The nucleic acid used according to the invention may be a DNA or RNAsequence, especially in a linear form. The nucleic acid may be isolatedfrom natural sources, for example, from eukaryotic tissues, preferablymammal tissues, more preferably from human tissues, or preparedsynthetically.

According to the invention, it is provided, in particular, that thenucleic acid used as the agent, if inserted in a vector, especially anexpression vector, can inhibit the expression of the gene of humanprotein kinase C-β in a host cell in antisense orientation to apromoter. When the nucleic acid employed according to the invention isinserted in a vector in antisense orientation, i.e., when an antisenseconstruct of the nucleic acid employed according to the invention isemployed, the nucleic acid will be transcribed as an antisense nucleicacid. Then, when the native PKC-β gene of the cell is transcribed, theantisense transcript produced of the nucleic acid used according to theinvention can bind to the mRNA transcript of the native protein kinaseC-β gene which is in sense orientation to form a duplex structure. Inthis way, the translation of the mRNA of the native PKC-β gene into apolypeptide is selectively suppressed, and the expression of the nativePKC-β is specifically inhibited without inhibiting the expression ofother cellular PKC isoforms.

In a preferred embodiment of the invention, it is provided that thenucleic acid used for the production of antisense constructs does notcomprise the entire sequence coding for PKC-β, but only fragmentsthereof. Such fragments comprise at least 10 nucleotides, preferably atleast 50 nucleotides, more preferably at least 200 nucleotides, whereinthe nucleotide regions of the sequence coding for PKC-β which arespanned by the fragments are selected in such a way that, when thefragments are expressed in antisense orientation in a cell, specificinhibition of the expression of PKC-β, especially human PKC-β, occurs,but not inhibition of other PKC isoforms.

According to the invention, it is provided that the above mentionednucleic acid or the suitable fragment thereof is inserted in a vectorunder the control of at least one expression regulating element, whereinthe nucleic acid or its fragment is inserted in an antisense orientationwith respect to said expression regulating elements. Thus, after thevector has been introduced into a cell, for example, a mammal cell,especially a human cell, the nucleic acid or its fragment can beexpressed in antisense orientation and thus efficiently inhibit theexpression of the native PKC-β of the cell. Preferably, the vector is aplasmid, cosmid, bacteriophage or virus.

Therefore, the present invention also relates to a vector whichcomprises a nucleic acid sequence coding for PKC-β or a fragment thereofunder the functional control of at least one expression regulatingelement, wherein the nucleic acid or its fragment is inserted in anantisense orientation with respect to said expression regulatingelement. Said expression regulating element is, in particular, apromoter, a ribosome binding site, a signal sequence or a 3′transcription terminator.

Another embodiment of the invention relates to a host cell whichcontains an above described vector. In particular, the host cell is amammal cell, preferably a human cell. In a particularly preferred form,the human cell is an adult stem cell.

In a preferred embodiment of the invention, synthetically preparedantisense oligonucleotides which comprise at least 10 nucleotides,preferably at least 50 nucleotides, more preferably at least 200nucleotides, are employed for inhibiting the expression of PKC-β. Suchantisense oligonucleotides can be directly employed for inhibiting PKC-βexpression, i.e., need not be inserted into a vector and expressed undercellular conditions.

In another preferred embodiment of the invention, it is provided that anantibody which specifically reacts with protein kinase C-β or a suitablefragment thereof is employed for inhibiting the activity of proteinkinase C-β. According to the invention, the antibody may be a monoclonalor polyclonal antibody. The antibody employed according to the inventionmay also be a humanized antibody.

In a further preferred embodiment of the invention, it is provided thata substance which changes the phosphorylation state of protein kinaseC-β is employed for inhibiting the activity of protein kinase C-β.

In another preferred embodiment of the invention, it is provided that aderivative or analogue of protein kinase C-β which acts as an antagonistof PKC-β is employed for inhibiting the activity of protein kinase C-β.Preferably, the derivatives or analogues of PKC-β employed according tothe invention are substances which compete with the native PKC-β for thebinding to PKC-β-specific substrates, but without causing the sameeffect as PKC-β after binding to the substrates.

In a particularly preferred embodiment of the invention, the compoundsas described in the documents U.S. Pat. No. 5,491,242, U.S. Pat. No.5,661,173, U.S. Pat. No. 5,481,003, U.S. Pat. No. 5,668,152, U.S. Pat.No. 5,672,618, WO 95/17182, WO 95/35294 and WO 02/ are employed forspecifically inhibiting and reducing the expression and/or activity ofprotein kinase C-β.

Another preferred embodiment of the invention relates to the use ofagents which specifically reduce or inhibit the expression and/oractivity of protein kinase C-α (PKC-α) for the preparation of apharmaceutical composition for the treatment and/or prevention ofcoronary heart disease, myocardial infarction, peripheral occlusivedisease, stroke, renal diseases involving proteinuria, diabetic lateeffects and/or cardiovascular complications in patients with diabetesmellitus, cardiovascular complications in patients with hypertension,and cardiovascular complications in patients with hypercholesterolemia.According to the invention, the cardiovascular complications arepreferably coronary heart disease, myocardial infarction, peripheralocclusive disease and stroke. The diabetic late effects are, inparticular, diabetic retinopathy, diabetic neuropathy and diabeticnephropathy.

In the context of the present invention, a “pharmaceutical composition”or a “medicament” means a mixture used for diagnostic, therapeuticand/or prophylactic purposes, i.e., a mixture which promotes or restoresthe health of a human or animal body, which comprises at least onenatural or synthetically prepared active ingredient which causes thetherapeutic effect. The pharmaceutical composition may be both a solidand a liquid mixture. For example, a pharmaceutical compositioncomprising the active ingredient may contain one or morepharmaceutically acceptable components. In addition, the pharmaceuticalcomposition may comprise additives usually employed in the art, forexample, stabilizers, production agents, separating agents,disintegrants, emulsifiers or other materials usually employed for thepreparation of pharmaceutical compositions.

According to the invention, in particular, the use of agents whichspecifically reduce or inhibit the expression and/or activity of proteinkinase C-α (PKC-α) as an active ingredient for the preparation of amedicament for the therapy and/or prophylaxis of the above mentioneddiseases is provided. In a preferred embodiment of the invention, theagents employed for the preparation of pharmaceutical compositions areselected from the group consisting of nucleic acids which reduce orinhibit the expression of the protein kinase C-α gene, vectorscontaining said nucleic acid, host cells containing said vectors,substances which inhibit or reduce the expression of protein kinase C-α,substances which inhibit the translocation of protein kinase C-α,antagonists of protein kinase C-α activity, and inhibitors of proteinkinase C-α activity.

More preferably, the agents employed for the preparation of thepharmaceutical composition according to the invention are antisenseoligonucleotides of the gene coding for protein kinase C-α, tocopherol,phorbol compounds, derivatives of protein kinase C-α, or analogues ofprotein kinase C-α.

In a preferred embodiment of the invention, it is provided that thepharmaceutical composition is used for parenteral, especiallyintravenous, intramuscular, intracutaneous or subcutaneousadministration. Preferably, the medicament containing the agentsemployed according to the invention is in the form of an injection orinfusion.

In another embodiment of the invention, it is provided that thepharmaceutical composition containing the agents employed according tothe invention is administered orally. For example, the medicament isadministered in a liquid dosage form, such as a solution, suspension oremulsion, or in a solid dosage form, such as a tablet.

Therefore, the present invention also relates to pharmaceuticalcompositions for the prevention and/or treatment of coronary heartdisease, myocardial infarction, peripheral occlusive disease, stroke,renal diseases involving proteinuria, diabetic late effects and/orcardiovascular complications in patients with diabetes mellitus,cardiovascular complications in patients with hypertension, andcardiovascular complications in patients with hypercholesterolemia,comprising at least one agent which specifically reduces or inhibits theexpression and/or activity of protein kinase C-α (PKC-α) as an activeingredient.

In a preferred embodiment, the agents contained in the pharmaceuticalcomposition are selected from the group consisting of nucleic acidswhich reduce or inhibit the expression of the protein kinase C-α gene,vectors containing said nucleic acid, host cells containing saidvectors, substances which inhibit or reduce the expression of proteinkinase C-α, substances which inhibit the translocation of protein kinaseC-α, antagonists of protein kinase C-α activity, and inhibitors ofprotein kinase C-α activity.

More preferably, the pharmaceutical composition according to theinvention contains antisense oligonucleotides of the gene coding forprotein kinase C-α, tocopherol, phorbol compounds, derivatives ofprotein kinase C-α, or analogues of protein kinase C-α.

In another preferred embodiment of the invention, the pharmaceuticalcomposition according to the invention contains at least one furtheractive ingredient. In particular, said further active ingredient is anagent which specifically reduces or inhibits the expression and/oractivity of protein kinase C-β.

Preferably, the agent which specifically reduces or inhibits theexpression and/or activity of protein kinase C-β is selected from thegroup consisting of nucleic acids which reduce or inhibit the expressionof the protein kinase C-β gene, vectors containing said nucleic acid,host cells containing said vectors, substances which inhibit or reducethe expression of protein kinase C-β, substances which inhibit thetranslocation of protein kinase C-β, antagonists of protein kinase C-βactivity, and inhibitors of protein kinase C-β activity.

Further advantageous embodiments of the invention can be seen from thedependent claims.

The invention is further illustrated by means of the following Figuresand Examples.

FIG. 1 shows the albumin excretion in the urine of PKC-alpha “knock out”mice with diabetes mellitus and without diabetes mellitus (control) andSV129 mice with diabetes and without diabetes (control). The albuminconcentration was determined by using an indirect ELISA assay. Thealbumin values established were based on the creatinin concentration.The non-diabetic SV129 and PKC-α^(−/−) mice have a comparablealbumin/creatinin quotient which is usually below 10 g/mol. In contrast,the quotient for diabetic SV129 mice is significantly higher (p=0,004).The values for the diabetic PKC-α^(−/−) mice are significantly lowerthan the values for the diabetic SV129 mice (p≦0.001). The transversebar indicates the median value. The significance was calculated by meansof the Mann-Whitney U test.

FIG. 2 shows the glomerular VEGF expression in PKC-alpha “knock out”mice with diabetes mellitus and without diabetes mellitus (control) andSV129 mice with diabetes and without diabetes (control). For each animalgroup, 40 glomeruli were evaluated semiquantitatively by means ofimmunohistochemical methods, and the values were divided into weak,medium and strong immunofluorescence. The significance was calculated bymeans of the Mann-Whitney U test. In diabetic animals, the VEGFexpression is significantly higher as compared to control animals(p<0.001). However, the VEGF expression in diabetic SV129 animals issignificantly higher as compared to diabetic PKC-alpha^(−/−) animals(p<0.001).

FIG. 3 shows the glomerular VEGF receptor II expression in PKC-alpha“knock out” mice with diabetes mellitus and without diabetes mellitus(control) and SV129 mice with diabetes and without diabetes (control).For each animal group, 40 glomeruli were evaluated semiquantitatively bymeans of immunohistochemical methods, and the values were divided intoweak, medium and strong immunofluorescence. The significance wascalculated by means of the Mann-Whitney U test. In diabetic animals, theVEGFR-II expression is significantly higher as compared to controlanimals (p<0.001). However, the VEGFR II expression in diabetic SV129animals is significantly higher as compared to diabetic PKC-alpha^(−/−)animals (p<0.001).

FIG. 4 shows the glomerular perlecan expression in PKC-alpha “knock out”mice with diabetes mellitus and without diabetes mellitus (control) andSV129 mice with diabetes and without diabetes (control). For each animalgroup, 40 glomeruli were evaluated semiquantitatively by means ofimmunohistochemical methods, and the values were divided into weak,medium and strong immunofluorescence. The significance was calculated bymeans of the Mann-Whitney U test. In diabetic SV129 animals, theperlecan expression is significantly lower as compared to SV129 controlanimals (p<0.001).

EXAMPLE 1

Experimental Diabetes Induction

With mice which were kept under standardized conditions at 22° C. withfree access to feed and water, the following experiments were performedafter approval by the animal protection authorities of Lower Saxony.

Before the start of the experiment, the blood sugar level from serum wasdetermined for all animals. The results are shown in Table 1. In 16SV129 control mice and 14 SV129 protein kinase C-alpha knock-out(PKC-α^(−/−)) mice, diabetes was induced by the injection ofstreptozotozin. Streptozotozin results in destruction of theinsulin-producing islet cells in the pancreas. The resulting insulindeficiency causes permanently increased blood sugar levels, i.e.,hyperglycemia, and thus diabetes mellitus. To produce the hyperglycemia,the animals were administered 125 mg each of streptozotozin per kg ofbody weight intraperitoneally on days 1 and 4. For such purposes, thestreptozotozin was dissolved in a 50 mM Na citrate solution with a pHvalue of 4.5. For control, seven SV129 and six PKC-α^(−/−) mice wereadministered only the solvent intraperitoneally on days 1 and 4.Subsequently, a drop of blood was taken from the tail of the mice everytwo days in order to check the blood sugar level. The blood sugarmeasurement was performed by means of the Bayer Glucometer Elite®measuring device. Glucometer Elite Sensor® test strips were used for thedetermination.

On days 7-10, the animals to which streptozotozin had been administeredwere diabetic with blood sugar levels of above 350 mg/dl. The startingvalues before streptozotozin injection were on average at 200 mg/dl.Animals which had obtained only the solvent did not exhibit any increaseof the blood sugar levels and did not develop diabetes mellitus. Tendays after the first injection, the animals were observed for further 8weeks. During this time, the blood sugar was checked every two weeks toensure that the diabetic animals were still diabetic. During thisperiod, the sugar levels varied on average around 500-550 mg/dl for thediabetic animals and around 200 mg/dl for the non-diabetic animals.

After 8 weeks, the animals were anesthetized with the narcotic avertin.Under anesthesia, 400 μl of blood was subsequently taken from the venousplexus of the eye, and the whole bladder urine was taken from thebladder by a puncture with a 27 G needle. Subsequently, the kidneys wereperfused with a Ringer lactate solution through the ventral aorta, andthe kidneys were removed. Immediately thereafter, the animals werekilled while under anesthesia. Subsequently, the blood sugar levels weredetermined from the serum. The blood sugar levels can be found inTable 1. As can be seen, the diabetic animals have about 2.5 to 3 timeshigher glucose levels than they had at the beginning of the experimentand also as compared with the non-diabetic control animals. TABLE 1Serum glucose in diabetic and non-diabetic mice before the beginning andat the end of the experiment Serum glucose before Serum glucose at thebeginning of the end of the the experiment (mg/dl) experiment (mg/dl)SV129 control (n = 7) 205 +/− 40 223 +/− 43 SV129 diabetic (n = 16) 192+/− 36  505 +/− 80* PKC-α^(−/−) control (n = 6) 223 +/− 27 197 +/− 21PKC-α^(−/−) diabetic (n = 14) 225 +/− 31  589 +/− 98**p ≦ 0.001 as compared to non-diabetic control animals

EXAMPLE 2

Determination of Albumin Concentration

The development of albuminuria in patients with diabetes is a knownphenomenon. Therefore, the albumin excretion in the urine of PKC-alpha“knock out” mice with diabetes mellitus and without diabetes mellitus(control) and SV129 mice with diabetes and without diabetes (control)was determined. For this purpose, the albumin concentration wasdetermined in the collected urine. To determine the albuminconcentration, an indirect ELISA assay (Albuwell M® of Exocell Inc.,Philadelphia, USA) was used. This ELISA assay is specific for murinealbumin. The determination was effected in accordance with themanufacturer's instructions. To be able to account for variations in theurine excretion, the albumin values determined were based on thecreatinin level in the urine. The results are shown in FIG. 1. It wasfound that the non-diabetic SV129 and PKC-α^(−/−) mice have a comparablealbumin/creatinin quotient which is usually below 10 g/mol. In contrast,the quotient for diabetic SV129 mice is significantly higher (p=0,004).The median value is 21.5 g/mol vs. 7.48 g/mol for non-diabetic SV129control animals. In comparison, there is no significant increase ofalbuminuria in diabetic PKC-α^(−/−) mice. The albumin/creatinin quotientis always below 20 g/mol, and the median is at 10.2 g/mol. The medianfor the non-diabetic PKC-α^(−/−) control mice is at 8.5 g/mol. Thevalues for the diabetic PKC-α^(−/−) mice is significantly lower than thevalues for the diabetic SV129 mice (p≦0.001). The results are shown inFIG. 1.

EXAMPLE 3

Determination of VEGF and VEGF Receptor II Expression

As set forth above in Example 1, all the animals were killed when theexperiment was over. Immediately thereafter, the kidneys were removedand frozen at −70° C. A further analysis of the removed kidneys showed asignificant increase of the expression of the “vascular endothelialgrowth factor” (VEGF) and VEGF receptor II (VEGFR-II) in the renalcorpuscles (glomeruli) of the diabetic control animals. The detection ofVEGF and VEGFR-II expression was effected by immunohistochemicalmethods. Thus, the kidneys frozen at −70° C. were cryo-sliced to athickness of 6 nm and then dried in air. Subsequently, the cryo-sliceswere fixed with cold acetone, dried in air and washed with tris buffer(TBS: 0.05 M tris buffer, 0.15 M NaCl, pH 7.6). The cryo-slices weresubsequently incubated for 60 minutes in a moist chamber with a primarypolyclonal “rabbit” antibody against murine VEGF (Santa Cruz, A-20) orVEGFR-II (Santa Cruz, C-1158). After washing anew with TBS, the sliceswere incubated with a Cy3-labeled secondary “anti-rabbit” antibody(Jackson Immunresearch Laboratories, 711-165-152) for 30 minutes at roomtemperature and again washed with TBS. Subsequently, the preparationswere evaluated and photographed through a Zeiss Axioplan-2 microscope(Zeiss, Jena, Germany). In all animals, 40 renal corpuscles each wereevaluated, and the fluorescence intensity was divided into strong,medium and weak. In diabetic SV129 control animals, a significantincrease (p<0.001) of VEGF and VEGFR-II expression was found as comparedto non-diabetic control animals. In comparison, the increase of theexpression was significantly less pronounced in diabetic SV129 andPKC-α^(−/−) mice (p<0.001). The results are shown in FIGS. 2 and 3.

EXAMPLE 4

Determination of Perlecan Expression

Since the established difference in VEGF expression alone could notaccount for the difference in albuminuria, the expression of the heparansulfate proteoglycan perlecan in the kidneys of diabetic andnon-diabetic animals was examined by means of immunohistochemicalmethods. Cryo-slices of the kidneys were prepared and embedded asdescribed in Example 3. A monoclonal rat antibody directed againstmurine perlecan (RDI Systems, A7L6) was used as the primary antibody. ACy3-labeled donkey anti-rat antibody (Jackson ImmunresearchLaboratories, 712-165-153) was used as the secondary antibody. Theimmunohistochemical examination of the slices gave the completelysurprising result that perlecan was no longer or hardly any longerdetectable in diabetic control animals (cf. FIG. 4). Thus, perlecancould be detected neither in the glomerulus nor in the vascular wall ofarterioles. In contrast, the expression of perlecan was unchanged or butslightly reduced in SV129 and PKC-α^(−/−) mice. Since the lack ofheparan sulfate is considered one of the main mediators in thedevelopment of proteinuria, it is to be considered that this result canaccount for the absence of albuminuria.

1. A method of treatment and/or prevention of vascular diseases,cardiovascular diseases, renal diseases involving proteinuria, diabeticlate effects and/or cardiovascular complications in patients withdiabetes mellitus, cardiovascular complications in patients withhypertension, and/or cardiovascular complications in patients withhypercholesterolemia, comprising administering at least one agent whichreduces or inhibits the expression and/or activity of protein kinase C-α(PKC-α).
 2. The method of claim 1, wherein said vascular diseases andcardiovascular diseases are selected from the group consisting ofperipheral occlusive disease, coronary heart disease, myocardialinfarction and stroke.
 3. The method of claim 1, wherein saidcardiovascular complications are selected from the group consisting ofperipheral occlusive disease, coronary heart disease, myocardialinfarction and stroke.
 4. The method of claim 1, wherein said diabeticlate effects are selected from the group consisting of diabeticretinopathy, diabetic neuropathy and diabetic nephropathy.
 5. The methodof claim 1, wherein said renal diseases involving proteinuria areparenchymal kidney diseases.
 6. The method of claim 5, wherein saidproteinuria is selected from the group consisting of glomerularproteinuria, tubular proteinuria and mixed glomerulo-tubularproteinuria.
 7. The method of claim 5, wherein said renal diseases areselected from the group consisting of minimal-change nephropathy, otherglomerulopathies, kidney amyloidosis, hereditary tubulopathy,renal-tubular azidosis, interstitial nephritis induced by bacteria ormedicaments, acute renal failure, Bence-Jones nephropathy and kidneytransplantation.
 8. (canceled)
 9. The method of claim 1, wherein saidagent is selected from the group consisting of at least one nucleic acidwhich reduces or inhibits the expression of the protein kinase C-α gene,a vector containing said nucleic acid, a host cell containing saidvector, a substance which reduces or inhibits the expression of proteinkinase C-α, a substance which inhibits the translocation of proteinkinase C-α, an antagonist of protein kinase C-α activity, and aninhibitor of protein kinase C-α activity.
 10. The method of claim 9,wherein said nucleic acid can inhibit the expression of the gene ofhuman protein kinase C-α in a host cell in anti-sense orientation to apromoter.
 11. The method of claim 9, wherein said nucleic acid isselected from the group consisting of a) a nucleic acid coding for humanprotein kinase C-α, or a fragment thereof; b) a nucleic acid which iscomplementary to the nucleic acid of group a), or a fragment thereof; c)a nucleic acid which is obtainable by substitution, addition, inversionand/or deletion of one or more bases of a nucleic acid of group a) orb), or a fragment thereof; and d) a nucleic acid which has more than 80%homology with a nucleic acid any of group a) through c), or a fragmentthereof.
 12. The method of claim 11, wherein said fragment of thenucleic acid of any of group a) through d) comprises at least 10nucleotides.
 13. The method of claim 9, wherein said nucleic acid is aDNA or a RNA.
 14. The method of claim 9, wherein said nucleic acid orfragment thereof is inserted in a vector under the control of at leastone expression regulating element in antisense orientation thereto. 15.The method of claim 14, wherein said vector is selected from the groupconsisting of a plasmid, a cosmid, a bacteriophage or a virus.
 16. Themethod of claim 14, wherein said expression regulating element isselected from the group consisting of a promoter, a ribosome bindingsite, a signal sequence or a 3′ transcription terminator.
 17. The methodof claim 14, wherein said vector is contained in a host cell.
 18. Themethod of claim 17, wherein said host cell is a mammalian cell.
 19. Themethod of claim 9, wherein said substance which inhibits or reduces theexpression of protein kinase C-α is an activator of protein kinase C-α.20. The method of claim 19, wherein said activator is a phorbolcompound.
 21. The method of claim 20, wherein said phorbol compound isselected from a group consisting of 12-O-tetradecanoylphorbol-13-acetate(TPA) and phorbol-12,13-dibutyrate (PDBu).
 22. The method of claim 9,wherein said inhibitor of protein kinase C-α activity is an antibodywhich reacts with protein kinase C-α.
 23. The method of claim 22,wherein said antibody is selected from a group consisting of amonoclonal antibody and a polyclonal antibody.
 24. The method of claim22, wherein said antibody is a humanized antibody.
 25. The method ofclaim 9, wherein said inhibitor of protein kinase C-α activity changesthe phosphorylation state of protein kinase C-α.
 26. The method of claim25, wherein said inhibitor is tocopherol.
 27. The method of claim 9,wherein said antagonist is selected from a group consisting of aderivative and an analogue of protein kinase C-α.
 28. The method ofclaim 1, wherein said agent which reduces or inhibits the expressionand/or activity of protein kinase C-α is an agent which reduces orinhibits the expression and/or activity of protein kinase C-β.
 29. Themethod of claim 28, wherein said agent is cyclosporine A.
 30. The methodof claim 1, wherein said agent which specifically reduces or inhibitsthe expression and/or activity of protein kinase C-α is administered incombination with an agent which reduces or inhibits the expressionand/or activity of protein kinase C-β.
 31. The method of claim 30,wherein said agent which reduces or inhibits the expression and/oractivity of protein kinase C-β is selected from the group consisting ofat least one nucleic acid which reduces or inhibits the expression ofthe protein kinase C-β gene, a vector containing said nucleic acid, ahost cell containing said vector, a substance which reduces or inhibitsthe expression of protein kinase C-β, a substance which inhibits thetranslocation of protein kinase C-β, an antagonist of protein kinase C-βactivity, and an inhibitor of protein kinase C-β activity.
 32. Themethod of claim 31, wherein said nucleic acid is selected from the groupconsisting of a) a nucleic acid coding for human protein kinase C-β, ora fragment thereof; b) a nucleic acid which is complementary to thenucleic acid of group a), or a fragment thereof; c) a nucleic acid whichis obtainable by substitution, addition, inversion and/or deletion ofone or more bases of a nucleic acid of group a) or b), or a fragmentthereof; and d) a nucleic acid which has more than 80% homology with anucleic acid of any of group a) through c), or a fragment thereof. 33.The method of claim 32, wherein said fragment of the nucleic acid of anyof group a) through d) comprises at least 10 nucleotides.
 34. The methodof claim 31, wherein said nucleic acid is a DNA or a RNA.
 35. The methodof claim 31, wherein said nucleic acid or fragment thereof is insertedin a vector under the control of at least one expression regulatingelement in antisense orientation thereto.
 36. The method of claim 35,wherein said vector is a plasmid, a cosmid, a bacteriophage or a virus.37. The method of claim 35, wherein said expression regulating elementis a promoter, a ribosome binding site, a signal sequence or a 3′transcription terminator.
 38. The method of claim 35, wherein saidvector is contained in a host cell.
 39. The method of claim 38, whereinsaid host cell is a mammalian cell.
 40. The method of claim 31, whereinsaid inhibitor of protein kinase C-β activity is an antibody whichreacts with protein kinase C-β.
 41. The method of claim 40, wherein saidantibody is a monoclonal or a polyclonal antibody.
 42. The method ofclaim 40, wherein said antibody is a humanized antibody.
 43. The methodof claim 31, wherein said inhibitor of protein kinase C-β activitychanges the phosphorylation state of protein kinase C-β.
 44. The methodof claim 31, wherein said antagonist is a derivative of protein kinaseC-β or an analogue of protein kinase C-β.
 45. A method of preparation ofa pharmaceutical composition for the treatment and/or prevention ofcoronary heart disease, myocardial infarction, peripheral occlusivedisease, stroke, renal diseases involving proteinuria, diabetic lateeffects and/or cardiovascular complications in patients with diabetesmellitus, cardiovascular complications in patients with hypertension,and cardiovascular complications in patients with hypercholesterolemia,comprising the use of at least one agent which reduces or inhibits theexpression and/or activity of protein kinase C-α (PKC-α).
 46. The methodof claim 45, wherein said cardiovascular complications are coronaryheart disease, myocardial infarction, peripheral occlusive disease orstroke.
 47. The method of claim 45, wherein said diabetic late effectsare diabetic retinopathy, diabetic neuropathy and diabetic nephropathy.48. The method of claim 45, wherein said agent is selected from thegroup consisting of a nucleic acid which reduces or inhibits theexpression of the protein kinase C-α gene, a vector containing saidnucleic acid, a host cell containing said vector, a substance whichreduces or inhibits the expression of protein kinase C-α, a substancewhich inhibits the translocation of protein kinase C-α, an antagonist ofprotein kinase C-α activity, and an inhibitor of protein kinase C-αactivity.
 49. The method of claim 48, wherein said agent is selectedfrom the group consisting of an antisense oligonucleotide of a genecoding for a protein selected from the group consisting of proteinkinase C-α, tocopherol, phorbol compounds, derivatives of protein kinaseC-α, and analogues of protein kinase C-α.
 50. A pharmaceuticalcomposition for the treatment and/or prevention of coronary heartdisease, myocardial infarction, peripheral occlusive disease, stroke,renal diseases involving proteinuria, diabetic late effects and/orcardiovascular complications in patients with diabetes mellitus,cardiovascular complications in patients with hypertension, andcardiovascular complications in patients with hypercholesterolemia,comprising at least one agent which reduces or inhibits the expressionand/or activity of protein kinase C-α (PKC-α) as an active ingredient.51. The pharmaceutical composition of claim 50, wherein said agent isselected from the group consisting of a nucleic acid which reduces orinhibits the expression of the protein kinase C-α gene, a vectorcontaining said nucleic acid, a host cell containing said vector, asubstance which reduces or inhibits the expression of protein kinaseC-α, a substance which inhibits the translocation of protein kinase C-α,an antagonist of protein kinase C-α activity, and an inhibitor ofprotein kinase C-α activity.
 52. The pharmaceutical composition of claim51, wherein said agent is selected from the group consisting of anantisense oligonucleotide of the gene coding for protein kinase C-α,tocopherol, a phorbol compound, a derivative of protein kinase C-α, andan analogue of protein kinase C-α.
 53. The pharmaceutical composition ofclaims 50, comprising at least one additional active ingredient.
 54. Thepharmaceutical composition of claim 53, wherein said additional activeingredient is an agent which reduces or inhibits the expression and/oractivity of protein kinase C-β.
 55. The pharmaceutical composition ofclaim 54, wherein said agent which reduces or inhibits the expressionand/or activity of protein kinase C-β is selected from the groupconsisting of a nucleic acid which reduces or inhibits the expression ofthe protein kinase C-β gene, a vector containing said nucleic acid, ahost cell containing said vector, a substance which inhibits or reducesthe expression of protein kinase C-β, a substance which inhibits thetranslocation of protein kinase C-β, an antagonist of protein kinase C-βactivity, and an inhibitor of protein kinase C-β activity.
 56. Themethod of claim 11, wherein said fragment of the nucleic acid of any ofgroup a) through d) comprises at least 50 nucleotides.
 57. The method ofclaim 11, wherein said fragment of the nucleic acid of any of group a)through d) comprises at least 200 nucleotides.
 58. The method of claim18, wherein said host cell is a human cell.
 59. The use according toclaim 32, wherein said fragment of the nucleic acid of any of group a)through d) comprises at least 50 nucleotides.
 60. The use according toclaim 32, wherein said fragment of the nucleic acid of any of group a)through d) comprises at least 200 nucleotides.
 61. The method of claim39, wherein said host cell is a human cell.